Soluble forms of hendra and nipah virus G glycoprotein

ABSTRACT

This invention relates to soluble forms of G glycoprotein from Hendra and Nipah virus. In particular, this invention relates to compositions comprising soluble forms of G glycoprotein from Hendra and Nipah virus and also to diagnostic and therapeutic methods using the soluble forms of G glycoprotein from Hendra and Nipah virus. Further, the invention relates to therapeutic antibodies including neutralizing antibodies, and vaccines for the prevention and treatment of infection by Hendra and Nipah viruses.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/629,682 (filed May 29, 2008) which is a U.S. National PhaseApplication of International PCT Application PCT/US05/24022 (filed Jul.7, 2005) which claims the benefit of U.S. Provisional Application60/586,843 (filed Jul. 9, 2004), all of which are herein incorporated byreference in their entirety.

RIGHTS IN THE INVENTION

This invention was made, in part, with support provided by the UnitedStates government under Grant No. U54 AI057168-01, awarded by theNational Institute of Allergy and Infectious Diseases, and Grant No.RO731L, awarded by the Uniformed Services University of the HealthSciences, and the United States government may have certain rights inthis invention.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled“044508-5022-SubstituteSequenceListing.txt” created on or about Jul. 9,2014 with a file size of about 17 kb contains the sequence listing forthis application and is hereby incorporated by reference in itsentirety.

1. FIELD OF THE INVENTION

This present invention relates to soluble forms of a G glycoprotein fromHendra and Nipah virus, to compositions comprising the soluble forms ofG glycoprotein from Hendra and Nipah virus, to antibodies reactiveagainst soluble forms of G glycoprotein from Hendra and Nipah virus, andto methods relating thereto.

2. DESCRIPTION OF THE BACKGROUND

Nipah virus and Hendra virus are emerging viruses that are responsiblefor previously unrecognized fatal diseases in animals and humans. Theseviruses are closely related members of a new genus, Henipavirus, in theParamyxoviridae family, a diverse group of large, enveloped,negative-sense RNA viruses, that includes a variety of important humanand animal pathogens. The recent emergence of these two viruses appearsto have been the result of exposure of new hosts precipitated by certainenvironmental and behavioral changes. Hendra virus was identified first,from cases of severe respiratory disease that fatally affected bothhorses and man. Subsequent to that appearance, an outbreak of severefebrile encephalitis associated with human deaths occurred in Malaysia.Later studies identified a Hendra-like virus, now known as Nipah virus,as the etiologic agent of that episode. These viruses are unusual amongthe paramyxoviruses in their abilities to infect and cause disease withhigh fatality rates in a number of host species, including humans, andare zoonotic Biological Safety Level-4 agents. Presently, the catappears to be the ideal small-animal model capable of reproducing thepathology seen in infected humans.

Nipah and Hendra virus are NIAID select, category C viruses and possessseveral features which make them highly adaptable for use as biowarfareagents. For example, both readily grow in cell culture or embryonatedchicken eggs, produce high un-concentrated titers near 1×10⁸ TCID₅₀/ml,(14), are highly infectious and transmitted via the respiratory tract(22, 27), and can be amplified and spread in livestock serving as asource for transmission to humans. Recent evidence also indicates thatnosocomial transmissibility of NiV from patients with encephalitis tohealthcare workers is possible (45, 60).

Fusion of the membrane of enveloped viruses with the plasma membrane ofa receptive host cell is a prerequisite for viral entry and infectionand an essential step in the life cycle of all enveloped viruses.Research towards dissecting and understanding the mechanisms of thisprocess is an important area of work. Not only does it afford insightsinto the complex interactions between viral pathogens and their hostcells, but it can also shed light on the complex and essentialbiochemical process of protein-mediated membrane fusion, and also leadto the development of novel intervention and vaccine strategies. Thishas been demonstrated in the HIV research field, where the discovery ofthe long-sought coreceptors involved in entry and infection has opened abroad new era in the development of therapeutics to block the infectionprocess at the level of entry (reviewed in (3, 18)).

Paramyxoviruses are negative-sense RNA enveloped viruses and encompass avariety of important human and animal pathogens, including measles virus(MeV), mumps virus, Sendai virus (SeV), Newcastle disease virus (NDV),rinderpest virus, canine distemper virus (CDV), human parainfluenzaviruses (hPIV) 1-4, respiratory syncytial virus (RSV), and simian virus5 (SV5) (reviewed in (36)). In contrast to retroviruses, paramyxovirusescontain two principal membrane-anchored glycoproteins, which appear asspikes projecting from the envelope membrane of the viral particle whenviewed under the electron microscope. One glycoprotein is associatedwith virion attachment to the host cell, and, depending on theparticular virus, has been designated as either thehemagglutinin-neuraminidase protein (RN), the hemagglutinin (H), or theG protein which has neither hemagglutinating nor neuraminidaseactivities (reviewed in (44)). The other glycoprotein is the fusionprotein (F) which is directly involved in facilitating the fusion of theviral and host cell membranes (reviewed in (36)). Following virusattachment to a permissive host cell, fusion at neutral pH (orindependently of the pH) between the virion and plasma membranes ensues,resulting in delivery of the nucleocapsid into the cytoplasm. In arelated process, cells expressing these viral glycoproteins on theirsurfaces can fuse with receptor-bearing cells, resulting in theformation of multinucleated giant cells (syncytia) under physiologicalor cell culture conditions.

The Envelope Glycoproteins.

The HN envelope glycoprotein is responsible for attachment of the virionto its receptor, sialic acid, on the target cell as is the case for thehPIVs, NDV, SV5 and others. In contrast, the morbilliviruses, like MeVand CDV, have an attachment protein (H) possessing only hemagglutinatingactivity and do not bind to sialic acid. MeV was the first morbillivirusshown capable of utilizing a cell-surface protein as a receptor (19,47), and was the demonstration of the predicted interaction between theMeV H glycoprotein and the MeV receptor CD46 using co-ip experiments andsoluble CD46 (48). In addition, MeV field isolates as well as vaccinestrains have been shown capable of utilizing signaling lymphocyteactivation molecule (SLAM; CD150) (61). SLAM is also capable of servingas a receptor for several other morbilliviruses, including CDV (62).

A third class of paramyxovirus attachment glycoproteins, which arepossessed by the Pneumovirinae such as RSV, are designated G, and haveneither hemmagglutinating nor neuraminidase activities (reviewed in(44)). The attachment glycoproteins are type II membrane proteins wherethe molecule's amino (N)-terminus is oriented towards the cytoplasm andthe protein's carboxy (C)-terminus is extracellular. The other majorenvelope glycoprotein is the fusion (F) glycoprotein, and the F of theseviruses are more similar, where in all cases it is directly involved inmediating fusion between the virus and host cell at neutral pH.

The F glycoprotein of the paramyxoviruses is a type I integral membraneglycoprotein with the protein's N-terminus being extracellular. Itshares several conserved features with other viral fusion proteins,including the envelope glycoprotein (Env) of retroviruses likegp120/gp41 of HIV-1, and hemagglutinin (HA) of influenza virus (reviewedin (26)). The biologically active F protein consists of two disulfidelinked subunits, F₁ and F₂/that are generated by the proteolyticcleavage of a precursor polypeptide known as F₀ (reviewed in (34, 55)).Likewise, HIV-1 Env and influenza HA are proteolytically activated by ahost cell protease, leading to the generation of a membrane distalsubunit analogous to F₂ and a membrane-anchored subunit analogous to F₁.In all cases, the membrane-anchored subunit contains a new N-terminusthat is hydrophobic and highly conserved across virus families and isreferred to as the fusion peptide (reviewed in (30)). Allparamyxoviruses studied to date require both an attachment and F proteinfor efficient fusion, with the exception of SV5 which can mediate somefusion in the absence of HN (50). Evidence of a physical associationbetween these glycoproteins has been observed with only limited successand only with NDV (57), hPIV (73), and recently with MeV (51), but theseobservations have often been with the aid of chemical cross-linkingagents. It is hypothesized that following receptor engagement, theattachment protein must somehow signal and induce a conformationalchange in F leading to virion/cell fusion (35, 53). That conformationaldistinctions existed in the HN and F of a paramyxovirus depending onwhether they were expressed alone or in combination has been noted forquite sometime (13).

The Paramyxovirus F envelope glycoproteins, like those of retroviruses,are considered class I membrane fusion-type proteins. An importantfeature of the fusion glycoproteins of these viruses is the presence of2 α-helical domains referred to as heptad repeats that are involved inthe formation of a trimer-of-hairpins structure during or immediatelyfollowing fusion (29, 56). These domains are also referred to as eitherthe amino (N)-terminal and the carboxyl (C)-terminal heptad repeats (orHR1 and HR2), and peptides corresponding to either of these domains caninhibit the activity of the viral fusion glycoprotein when presentduring the fusion process, first noted with sequences derived from thegp41 subunit of HIV-1 envelope glycoprotein (32, 67). Indeed, HIV-1fusion-inhibiting peptides have met with clinical success and are likelyto be the first approved fusion inhibitor therapeutics. Peptidesequences from either the N or C heptads of the F of SV5, MeV, RSV,hPIV, NDV, and SeV have also been shown to be potent inhibitors offusion (33, 37, 52, 68, 74, 75). It is generally accepted thatsignificant conformational change would occur during activation ofparamyxovirus F fusogenic activity. Differential antibody bindingreactivities of precursor and proteolytically processed forms of SV5 F(20) and in conjunction with the structure of the post fusion 6-helixbundle of SV5 F (2), strongly support the conformational change model,not only from the pre-fusion to post-fusion structural change, but alsofrom the F₀ precursor to the F₂-F₁ mature protein. That the post-fusionstructure of a paramyxovirus F core is likely conserved across otherparamyxoviruses has been further supported by the F core structures ofRSV (79) and MeV (80). However, recent structural studies on the Fglycoprotein of NDV have yielded some different and interestingfindings. The oligomeric trimer structure of NDV F (in perhaps thepre-fusion or meta-stable state) has offered some alternativeinformation which distinguishes it from the classic influenza HAstructure, this is principally reflected in the completely oppositeorientation of the central coiled coils formed by the HR1 (also termedHRA) segments of the trimer (9, 10). To date this is the only structuralinformation on the pre-fusion (or meta-stable) form of a paramyxovirus F(in fact the only other meta-stable, class I, structure other thaninfluenza HA), and perhaps represents a possible third-class of viralfusion proteins.

A precise understanding of how the fusion and attachment glycoproteinsfunction in concert in mediating fusion has yet to be elucidated, butthere are two central models proposed for the role of the attachmentglycoprotein in the paramyxovirus-mediated membrane fusion process,which were recently detailed by Morrison and colleagues (41), in thecontext of the HN glycoprotein of NDV. In the first model, the fusionand attachment glycoproteins are not physically associated in themembrane, but following receptor engagement there is an alteration inthe attachment glycoprotein which facilitates its association with F andin so doing imparts or facilitates F conformational change leading tomembrane fusion. In the second model, the F and attachment glycoproteinare pre-associated and receptor engagement induces conformationalalteration in the attachment glycoprotein, and this process alters orreleases an interaction with F that allows F to proceed towards itsfusion active state-formation of the 6-helix bundle just prior orconcomitant with membrane merger. Findings on NDV demonstrate thevariable accessibility of the HR1 domain during the process, where HR1of F are accessible to specific fusion-inhibiting antibody when F ispresented in the context of HN, however expression of F alone results ina non-fusogenic version of F with distinctly altered conformation havingan HR1 domain which is no longer accessible to antibody (41). The secondmodel is that the attachment glycoprotein is holding F in itsnon-fusogenic conformation and upon receptor engagement andconformational change in the attachment glycoprotein F is released toundergo conformational changes leading to 6-helix bundle formation andfacilitation of membrane fusion. This is supported by observations thatparamyxovirus F expressed alone neither mediates fusion (with theexception of SV5 under certain conditions) and has variably antibodyaccessibility of certain domains such as the NDV F HR1 domain (41). Thisis perhaps because F alone has transitioned to a fusion triggered orintermediate conformation at an inappropriate moment, which would beconsistent with observations of fusion defective or triggered HIV-1 gp41also referred to as dead-spikes (17). Preliminary findings with HeV andNiV support this second model. Finally, that the attachment glycoproteinof a paramyxovirus undergoes specific conformation alteration when boundto receptor has been recently revealed at the molecular level fromstudies on the HN glycoprotein of NDV (58, 59). These studies haverevealed clear differences in the structure of HN when thereceptor-bound glycoprotein is compared to the non-receptor-bound HNstructure. In addition, all known viral envelope glycoproteins are homo-or heterooligomers in their mature and functional forms (reviewed in(16)). Multimeric proteins, like these, generally interact over largeareas, making structural differences between monomeric subunits and themature oligomer likely (31). This feature can also translate intodifferences in antigenic structure and has been shown for a number ofproteins, most notably the trimeric influenza HA glycoprotein (69) andHIV-1 gp120/gp41 (7). Indeed, a trimer-specific, potent neutralizingdeterminant has been mapped to the interface between adjoining subunitsof HA, and oligomer-specific anti-HIV-1 Env antibodies have beenidentified and mapped to conformation-dependent epitopes in gp41 (7).Thus far, all paramyxoviruses, retroviruses, and influenza virus fusionglycoproteins appear to be homotrimers (8, 9, 21, 54, 71), and severalHN attachment proteins have been shown to be tetrameric, comprised of adimer of homodimers. For example, the NDV HN can form dimers andtetramers on the viral surface (40, 43), and recently the crystalstructure of the globular head region of the HN dimer from NDV has beensolved (15). Finally, and of importance in understanding certain aspectsof the immune response to these viruses and the development of vaccines,it is these major envelope glycoproteins of these viruses to whichvirtually all virus-neutralizing antibodies are directed against.

Emerging Pathogenic Paramyxoviruses.

In 1994, a new paramyxovirus, was isolated from fatal cases ofrespiratory disease in horses and humans, and was shown to be distantlyrelated to MeV and other members of the morbillivirus genus; it wasprovisionally termed equine morbillivirus (EMV) but has since beenre-named Hendra virus (HeV) (46). The first outbreak of severerespiratory disease in the Brisbane suburb of Hendra Australia resultedin the death of 13 horses and their trainer, and the non-fatal infectionof a stable hand and a further 7 horses. At approximately the same time,in an unrelated incident almost 100 km north of Hendra, a 35-year-oldman experienced a brief aseptic meningitic illness after caring for andassisting at the necropsies of two horses subsequently shown to havedied as a result of HeV infection. Thirteen months later this individualsuffered a recurrence of severe encephalitis characterized byuncontrolled focal and generalized epileptic-activity. A variety ofstudies that were performed in the evaluation of this fatality,including serology, PCR, electron microscopy (EM) andimmunohistochemistry, strongly suggested that HeV was indeed the causeof this patient's encephalitis, and the virus was acquired from theHeV-infected horses 13 months earlier (49). In all, fifteen horses andtwo people died in the two episodes. At the time the source of theemerging virus was undetermined, but more recently it has been foundthat approximately 50% of certain Australian fruit bat species, commonlyknown as flying foxes, have antibodies to HeV and Hendra-like viruseshave been isolated from bat uterine fluids. It appears that theseanimals are the natural host for the virus (22, 24, 25, 76). Recently,the nucleic acid sequence of HeV genes has been analyzed and comparedwith those of other paramyxoviruses (64, 77, 78). These studies haveconfirmed that HeV is a member of the Paramyxoviridae, subfamilyPararnyxovirinae.

Subsequent to these events, an outbreak of severe encephalitis in peoplewith close contact exposure to pigs in Malaysia and Singapore occurredin 1998 (1). The outbreak was first noted in September 1998 and bymid-June 1999, more than 265 cases of encephalitis, including 105deaths, had been reported in Malaysia, and 11 cases of disease with onedeath reported in Singapore. This outbreak had a tremendous negativeeconomic impact, which continues to date. Although successful, measurestaken in the early days of the outbreak resulted in the slaughter ofapproximately 1.3 million pigs and the virtual closure of the pigfarming industry in peninsular Malaysia. EM, serologic, and geneticstudies have since indicated that this virus is also a paramyxovirus,and was closely related to HeV. This virus was named Nipah virus (NiV)after the small town in Malaysia from which the first isolate wasobtained from the cerebrospinal fluid of a fatal human case (11, 12, 23,38, 39).

Most human patients present with acute encephalitis, which in theMalaysia outbreak of 1998-1999 ultimately resulted in a mortality rateof approximately 40%, but infection can also present as anonencephalitic or asymptomatic episode with seroconversion.Interestingly, infection with NiV can also take a more chronic coursewith more serious neurological disease occurring late (greater than 10weeks) following a nonencephalitic or asymptomatic infection. On theother hand, the recurrence of neurological manifestations (relapsedencephalitis) has also been noted in patients who had previouslyrecovered from acute encephalitis. Cases of relapsed-encephalitispresented from several months to nearly two years after the initialinfection (72) Taken together, there was nearly a 10% incidence rate oflate encephalitic manifestations with a mortality rate of 18%. Thus,with both NiV and HeV a prolonged period of infection is possible beforeserious neurological disease occurs. The underlying mechanisms whichallow these viruses, especially NiV, to escape immunological clearancefor such an extended period are completely unknown.

In the case of NiV, the late presentation of neurological disease andIgG subclass response showed similarities to subacute sclerosingpanencephalitis (SSPE), a rare late manifestation of MeV infection (72).It was molecular characterization of HeV and NiV which distinguishedthem as distinctly new paramyxoviruses. The families Paramyxoviridae,Filoviridae, Rhabdoviridae, and Bornaviridae are all negative-sense RNAenveloped viruses sharing similar genome organization, replicationstrategies, and polymerase domain structure (63). These families aregrouped in the order Mononegavirales, the first taxon above family levelvirus taxonomy. The genome size in the Mononegavirales is wide ranging,˜8.9-19.1 kb. The genomes of paramyxoviruses, as a group, are generallyconsidered tightly spread, having sizes in the range of 15.1-15.9 kb,except HeV and NiV whose genomes of 18.2 kb, far closer in size to theFiloviridae. Much of this added length is untranslated regions at the 3′end in the six transcription units, again quite similar to Marburg andEbloa Filoviruses (63). Also, the P protein is larger by 100 residues(longest known), and a small basic protein (SB) in HeV of unknownfunction. Taken together, the molecular features of both HeV and NiVmake them unusual paramyxoviruses, as does their ability to causepotentially fatal disease in a number of species, including humans.

Pathogenesis.

The development or characterization of animal models to study thesenewly identified viral zoonoses is important for understanding theirpathogenic features and in the development of therapeutics. Of the twofatal cases of HeV infection in humans, the first was the result ofsevere respiratory disease following several days of ventilatedlife-support. The patient's lungs had gross lesions of congestion,hemorrhage and oedema associated with histological chronic alveolitiswith syncytia. The second fatal case was one of leptomeningitis withlymphocytes and plasma cells and foci of necrosis in various parts ofthe brain parenchyma, after initial infection more than 1 yearpreviously (reviewed in (27)). Multinucleate endothelial cells were alsoseen in the viscera as well as in the brain. In contrast, there weremany more human cases of infection with NiV. More than 30 individualsresulting from the large NiV outbreak in Malaysia and Singapore wereautopsied, and the immuno- and histological features included systemicendothelial infection accompanied by vasculitis, thrombosis, ischaemiaand necrosis (reviewed in (27)). These changes were especially noted inthe central nervous system (CNS). Immunohistochemical analysis have alsoshown widespread presence of NiV antigens in neurons and otherparenchymal cells in necrotic foci seen in the CNS as well as inendothelial cells and media of affected vessels (27). In infectedhumans, evidence of vasculitis and endothelial infection was also seenin most organs examined. Disseminated endothelial cell infection,vasculitis, and CNS parenchymal cell infection play an essential role inthe fatal outcome of NiV infection in humans (reviewed in (27)). Theprincipal zoonotic episodes in nature involved the horse in the HeVcases and the pig in the case of NiV. Both these viruses have a notablebroad host cell tropism in vitro studies (4, 5). These observationscorrelated to what has been observed in natural and experimentalinfection.

Experimental infections of the horse and pig have been carried out withHeV and NiV respectively and one naturally NiV-infected horse has beenexamined. The pathology caused by either virus in horses appears to bemore severe than that caused by NiV in pigs. In addition to pigs, HeVinfection of cats has also been performed and in this case diseaseresembles that seen in horses, characterized by generalized vasculardisease with the most severe effects seen in the lung (28). Guinea pigshave also been experimentally infected with HeV (28) and the pathologyseen differed significantly in several respects in comparison to thehuman cases as well as natural and experimentally infected horses. Inguinea pigs HeV caused generalized vascular disease but, unlike horsesand cats, there was little or no pulmonary oedema. Histologically,vascular disease was prominent in arteries and veins, and in many organssuch as the lung, kidney, spleen, lymph nodes, gastrointestinal tractand skeletal and intercostal muscles. NiV infection of the guinea pighas not yet been well described.

In regards to other small laboratory animal models, NiV and HeV do notcause disease in mice even after subcutaneous administration, however,and not surprisingly, they will kill mice if administeredintracranially. Further, there is also no serological evidence for NiVin rodents in Malaysia, and several hundred sera were tested during theoutbreak. Evidence of natural NiV infections were also noted in dogs andcats.

In experimental NiV infection of the cat, gross lesions in animals withsevere clinical signs strongly resembled those of cats infected withHeV. These consisted of hydrothorax, oedema in the lungs and pulmonarylymph nodes, froth in the bronchi, and dense purple-red consolidation inthe lung. There were also similar features in the histologicalappearance, diffuse perivascular, peribronchial and alveolar hemorrhageand oedema, vasculitis affecting arteries and arterioles, alveolitis,syncytium formation within endothelial cells and alveolar epithelialcells (reviewed in (27)). Taken together, the evidence to date indicatesthat the cat represents an animal model whereby the pathology seen mostclosely resembles the lethal human disease course. In addition,infection of cats with either NiV or HeV is uniformly fatal. NiV and HeVappear to cause similar diseases but with some notable variations, andalthough the basic pathologic processes have been well described, lessis known about the factors which clearly influence disease coursedepending on the species infected. This is a special concern in humaninfections, where there is a remarkable ability of these viruses topersist in the host (up to 2 yrs) before causing a recurrence of severeand often fatal disease. Cats succumb within 6-8 days to subcutaneousinfection with 5,000, and subcutaneous or oral administration of 50,000,TCID₅₀ of a low passage, purified HeV (65, 66, 70). Experimentalinfection of cats with NW has confirmed the susceptibility of thisspecies to oronasal infection with 50,000 TCID₅₀ NiV (42). In summary,the clinical and pathological syndrome induced by NiV in cats wascomparable with that associated with HeV infection in this species,except that in fatal infection with NiV there was extensive inflammationof the respiratory epithelium, associated with the presence of viralantigen.

In summary, recurrent outbreaks of NiV resulting in significant numbersof human fatalities have recently been confirmed (Fatal fruit bat virussparks epidemics in southern Asia. Nature 429, 7, 06 May 2004). HeV isalso know to cause fatalities in human and animals and is geneticallyand immunologically closely related to NiV. There are presently novaccines or therapeutics for prevention of infection or disease causedby Nipah virus or Hendra virus. Both Nipah virus and Hendra virus areUnited States, National Institute of Allergy and Infectious Disease,category C priority agents of biodefense concern. Further, as theseviruses are zoonotic Biological Safety Level-4 agents (BSL-4),production of vaccines and/or diagnostics, with safety is very costlyand difficult. Thus, there is a need for a Nipah virus or Hendra virusvaccines and diagnostics that allow for high throughput production ofvaccines and/or diagnostics. All references cited herein, includingpatent applications and publications, are incorporated by reference intheir entirety.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides new toolsand methods for the design, production and use of soluble form's of theG envelope glycoprotein of Hendra virus and Nipah virus.

One embodiment of the invention is directed to polynucleotides andpolypeptides or fragments thereof encoding a soluble G protein derivedfrom Hendra virus.

Another embodiment of the invention is directed to polynucleotides orpolypeptides or fragments thereof encoding a soluble G protein derivedfrom Nipah virus.

Another embodiment is directed to methods of producing soluble G proteinderived from Hendra virus and/or Nipah virus.

Another embodiment is directed to expression vectors comprising thepolynucleotides encoding a soluble G protein derived from Hendra and/orNipah virus.

Another embodiment is directed to a fusion protein comprising apolypeptide of the invention and one or more other polypeptides thatenhance the stability of a polypeptide of the invention, enhance theimmunogenicity of a polypeptide of the present invention and/or assistin the purification of a polypeptide of the present invention.

Another embodiment is directed to antibodies and fragments thereof, suchas neutralizing antibodies, specific for a soluble G protein derivedfrom Hendra and/or Nipah virus and diagnostic and/or therapeuticapplication of such antibodies.

Another embodiment is directed to subunit vaccine comprising thepolynucleotides or polypeptides of the invention.

Another embodiment of the invention is directed to methods of preventinginfection with Hendra and/or Nipah virus in a subject or mitigating aninfection of Hendra and/or Nipah virus in a subject.

Another embodiment of this invention is directed to diagnostic kitscomprising the polynucleotides, polypeptides and/or antibodies of theinvention.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE FIGURES AND TABLE

FIG. 1 shows expression of soluble HeV G envelope glycoprotein. Vacciniavirus encoding either a myc-tag or S-tag soluble HeV G was produced bymetabolic labeling in HeLa cells. Control is wild-type HeV G. Specificprecipitation of each sG construct is shown by precipitation from eitherlysates or supernatants using myc MAb or S-beads.

FIG. 2 shows inhibition of HeV and NiV-mediated fusion by sG S-tag. HeLacells were infected with vaccinia recombinants encoding HeV F and HeV Gor NiV F and NiV G glycoproteins, along with a vaccinia recombinantencoding T7 RNA polymerase (effector cells). Each designated target celltype was infected with the E. coli LacZ-encoding reporter vaccinia virusvCB21R. Each target cell type (1×10⁵) was plated in duplicate wells of a96-well plate. sG S-tag or control supernatants were added and incubatedfor 30 minutes at 37° C. The HeV or NiV glycoprotein-expressing cells(1×10⁵) were then mixed with each target cell type. After 2.5 hr at 37°C., Nonidet P-40 was added and β-Gal activity was quantified. Panels Aand B: Inhibition of HeV-mediated fusion by sG S-tag-infectedsupernatant or WR-infected supernatant in U373 cells (Panel A) or PCI 13cells (Panel B). Panels C and D: Inhibition of HeV and NiV-mediatedfusion by sG S-tag in U373 cells (Panel C) or PCI 13 cells (Panel D).Panels E and F: Inhibition of HeV- and NiV-mediated fusion by purifiedsG myc-tag in U373 cells (Panel E) or PCI 31 cells (Panel F).

FIG. 3 shows indirect immunofluorescence of permissive andnon-permissive cell lines stained with sHeV G envelope glycoprotein.Cells were plated into 8 well Lab-Tek II chamber slides in theappropriate medium and incubated for 3 days. The cells were fixed withacetone for 2 minutes. HeLa cells represent a fusion non-permissive cellline whereas U373, PCI 13, and Vero represent fusion permissive celllines. The cells were stained with sG S-tag followed by an anti-HeV Gspecific rabbit antiserum and a donkey anti-rabbit Alexa Fluor 488conjugate. Samples were examined with an Olympus microscope with areflected light fluorescence attachment and an Olympus U-M41001 filter.All images were obtained with a SPOT RT CCD digital camera at anoriginal magnification of 40×. Panel A: sG S-tag and donkey anti-rabbitAlexa Fluor 488 conjugate. Panel B: sG S-tag, anti-HeV G antiserum, anddonkey anti-rabbit Alexa Fluor 488 conjugate.

FIG. 4 shows expression of soluble NiV G envelope glycoprotein. Vacciniavirus encoding S-tag soluble HeV G, or plasmid expression vectorsencoding S-tag soluble HeV G or NiV G were produced by metaboliclabeling in HeLa cells. Specific precipitation of each sG construct isshown by precipitation from either lysates or supernatants usingS-beads.

FIG. 5 shows analysis of the oligomeric structures of soluble HeV Genvelope glycoprotein. HeLa cells were infected with sHeV G (S-tag)encoding vaccinia virus and incubated 16 h at 37° C. (4 wells of a 6well plate). Beginning at 6 h post-infection, the cells weremetabolically labeled overnight with [35S]-met/cys. Supernatants wereremoved, clarified by centrifugation, concentrated, buffer exchangedinto PBS. One half (400 μl) of the sHeV G was then cross-linked withDTSSP [3,3′-Dithiobis(sulfosuccinimidylpropionate)] (4 mM/RT°/15 min)quenched with 100 mM Tris pH 7.5. The cross-linked and un-cross linkedpreparations were then divided into two equal portions and layered ontocontinuous (5-20%) sucrose gradients (4 gradients) and fractioned. Allfractions were then precipitated with S-protein agarose, and the samplesof metabolically labeled sG were resolved by 10% SDS-PAGE under reducingand non-reducing conditions and detected by autoradiography.

FIG. 6 shows a schematic of soluble Hey G glycoprotein constructs.IgK-chain linker (SEQ ID NO. 10); 15 aa linker (SEQ ID NO. 11);S-peptide tag (SEQ ID NO. 12); c-myc-epitope tag (SEQ ID NO 13); 15 aalinker (SEQ ID NO. 14).

FIG. 7 shows a molecular weight profile of HeV sG. A panel of highmolecular weight standards was separated on a Superdex 200 sizeexclusion column and a calibration curve was generated. Samples ofpurified sG_(S-tag) and sG_(myc-tag) were separated on the calibratedSuperdex 200 column and fractionated. The Kay values of major sG peakswere calculated and their apparent molecular weights were determinedusing the calibration curve from the molecular weight standards. Thefigure shows the three principal peaks observed with sG_(S-tag). Themolecular estimates shown associated with each of the three peaks (peak1, 2 and 3) are the averages of seven independent separations of threedifferent sG_(S-tag) preparations.

FIG. 8 shows oligomeric forms of sG_(S-tag). HeLa cells were infectedwith sG_(S-tag) encoding vaccinia virus and incubated 16 h at 37° C.Beginning at 6 h post-infection, the cells were metabolically labeledovernight with {³⁵S}-methionine/cysteine. Supernatants were removed,clarified by centrifugation, concentrated, buffer exchanged into PBS.One half (200 μl) of the sG_(S-tag) was then cross-linked with DTSSP[3,3′-Dithiobis(sulfosuccinimidylpropionate)] (4 mM/RT°/30 min) quenchedwith 100 mM Tris pH 7.5. The cross-linked and un-cross linkedpreparations were layered onto continuous (5-20%) sucrose gradients (2gradients) and fractioned. All fractions were split into 2 tubes (forreducing and non-reducing conditions), fractions were then precipitatedwith S-protein agarose, and the samples of metabolically labeled sG wereresolved by 7.5% SDS-PAGE under reducing and non-reducing conditions anddetected by autoradiography. Bottom and top of gradients are indicated.A: non-cross-linked and unreduced, B: non-cross-linked and reduced, C:cross-linked and unreduced, D: cross-linked and reduced.

FIG. 9 shows the oligomeric form of full length HeV G. HeLa cells wereinfected with HeV G encoding vaccinia virus and incubated 16 h at 37° C.Beginning at 6 h post-infection, the cells were metabolically labeledovernight with {³⁵S}-methionine/cysteine. Supernatants were removed andcells were chased for 2 h in complete medium, washed twice in PBS andrecovered. The non-cross-linked HeV G-expressing cells were lysed inTriton-X containing buffer, clarified by centrifugation, and layeredonto a continuous sucrose gradient (5-20%) and fractioned. All fractionswere split into 2 tubes (for reducing and non-reducing conditions),fractions were then precipitated with anti-HeV antiserum followed byProtein G-Sepharose, and the samples of metabolically labeled HeV G wereresolved by 7.5% SDS-PAGE under reducing and non-reducing conditions anddetected by autoradiography. Bottom and top of gradients are indicated.A: non-cross-linked and unreduced, B: non-cross-linked and reduced.

FIGS. 10 A, B, C and D shows immunofluorescence-based syncytia assay ofHeV and NiV. Vero cells were plated into 96 well plates and grown to 90%confluence. Cells were pretreated with sG_(S-tag) for 30 min at 37° C.prior to infection with 1.5×10³ TCID₅₀/ml and 7.5×10² TCID₅₀/ml of liveHeV or NiV (combined with sG_(S-tag)). Cells were incubated for 24hours, fixed in methanol and immunofluorescently labeled for P proteinprior to digital microscopy. Images were obtained using an Olympus IX71inverted microscope coupled to an Olympus DP70 high resolution colorcamera and all images were obtained at an original magnification of 85×.Representative images of FITC immunofluorescence of anti-P labeled HeVand NR. FIG. 10A: untreated control infections, FIG. 10B: infections inthe presence of 100 μg/ml sG_(S-tag).

FIG. 11 shows inhibition of HeV and NiV infection by sG_(S-tag). Verocells were plated into 96 well plates and grown to 90% confluence. Cellswere pretreated with sG_(S-tag) for 30 min at 37° C. prior to infectionwith 1.5×10³ TCID₅₀/ml and 7.5×10² TCID₅₀/ml of live HeV or NiV(combined with sG_(S-tag)). Cells were incubated for 24 hours, fixed inmethanol and immunofluorescently labeled for P protein prior to digitalmicroscopy and image analysis to determine the relative area of eachsyncytium. Figure shows the relative syncytial area (pixel²) versussG_(S-tag) concentration for HeV (circles) and NiV (triangles).

FIG. 12 shows the linear structure of the sG protein.

FIG. 13 shows a three dimensional structure of the sG protein.

FIG. 14 shows the amino acid sequence of the sG protein (SEQ ID NO 15).

Table 1 shows neutralization of HeV and NiV infection. Anti-HeV Gantisera were generated in rabbits by 3 inoculations with purifiedsG_(S-tag). Sera collected 2 weeks after the third injection wereanalyzed in a virus-neutralization assay against HeV and NiV. Serumneutralization titers were determined by presence of CPE (indicated by+) and recorded as the serum dilution where at least one of theduplicate wells showed no CPE.

DESCRIPTION OF THE INVENTION General Techniques

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, for example, MolecularCloning: A Laboratory Manual, second edition (Sambrook, et al., 1989)Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Methods in Molecular Biology, Humana Press; Cell Biology: ALaboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; AnimalCell Culture (R. I. Freshney, ed., 1987); Introduction to Cell andTissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Celland Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths,and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology(Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weirand C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols inMolecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: ThePolymerase Chain Reaction, (Mullis, et al., eds., 1994); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean,eds., Oxford University Press, 2000); Using antibodies: a laboratorymanual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press,1999); The Antibodies (M. Zanetti and J. D. Capra, eds., HarwoodAcademic Publishers, 1995).

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. For example, “a” G glycoproteinincludes one or more G glycoproteins.

Generally this invention provides soluble forms of HeV and NiV Gglycoprotein envelope protein, the polynucleotides encoding the proteinsand to methods for using these proteins in diagnosis, detection andtreatment. Specifically this invention provides soluble forms of HeV andNiV G glycoprotein envelope proteins which retain characteristics of thenative viral G glycoprotein allowing for rapid high throughputproduction of vaccines, diagnostics and screening.

Generally, the soluble forms of the HeV and NiV G glycoproteins compriseall or part of the ectodomain (e.g. extracellular) of the G glycoproteinof a HeV or NiV and are generally produced by deleting all or part ofthe transmembrane domain of the G glycoprotein and all or part of thecytoplasmic tail of the G glycoprotein. By way of example, a soluble Gglycoprotein may comprise the complete ectodomain of an HeV or NiV Gglycoprotein. Also by way of example, and not limitation a soluble Gglycoprotein may comprise all or part of the ectodomain and part of thetransmembrane domain of an HeV or NiV G glycoprotein.

The soluble HeV or NiV G glycoproteins of the invention, generallyretain one or more characteristics of the corresponding native viralglycoprotein, such as, ability to interact or bind the viral host cellreceptor, can be produced in oligomeric form or forms, or the ability toelicit antibodies (including, but not limited to, viral neutralizingantibodies) capable of recognizing native G glycoprotein. Examples ofadditional characteristics include, but are not limited to, the abilityto block or prevent infection of a host cell. Conventional methodologymay be utilized to evaluate soluble HeV or NiV G glycoproteins for oneof more of the characteristics. Examples of methodology that may be usedinclude, but are not limited to, the assays described herein in theExamples.

Polynucleotides

The term polynucleotide is used broadly and refers to polymericnucleotides of any length (e.g., oligonucleotides, genes, smallinhibiting RNA, fragments of polynucleotides encoding a protein etc). Byway of example, the polynucleotides of the invention may comprise all orpart of the ectodomain or all or part of the ectodomain and part of thetransmembrane domain. The polynucleotide of the invention may be, forexample, linear, circular, supercoiled, single stranded, doublestranded, branched, partially double stranded or single stranded. Thenucleotides comprising the polynucleotide may be naturally occurringnucleotides or modified nucleotides. Generally the polynucleotides ofthe invention encode for all or part of the ectodomain (e.g.extracellular) of the G glycoprotein of a HeV or NiV.

Non-limiting examples of sequences that may be used to construct asoluble HeV G glycoprotein can be found in Wang, L. F. et al., J. Virol.74 (21), 9972-9979 (2000) and Yu, M. et al., Virology 251 (2), 227-233(1998) (herein incorporated by reference in their entirety).Non-limiting examples of sequences that may be used to construct asoluble NiV G glycoprotein can be found in Harcourt, B H et al.,Virology 271: 334-349, 2000 and Chua, K. B. et al, Science 288 (5470),1432-1 (herein incorporated by reference in their entirety). Generally,G glycoprotein sequences from any Hendra virus and Nipah virus isolateor strain may be utilized to derive the polynucleotides and polypeptidesof the invention.

By way of example, and not limitation, a polynucleotide encoding asoluble HeV G Glycoprotein may comprise a polynucleotide sequenceencoding about amino acids 71-604 of the amino acid sequence for an HeVG Glycoprotein in Wang, L. F. et al., J. Virol. 74 (21), 9972-9979(2000) (SEQ ID NO: 16) (see also, e.g., Yu, M. et al., Virology 251 (2),227-233 (1998)). Also by way of example, and not limitation, apolynucleotide encoding a soluble HeV G glycoprotein may comprisenucleotides 9048 to 10727 of the polynucleotide sequence for an HeV Gglycoprotein in Wang, L. F. et al., J. Virol. 74 (21), 9972-9979 (2000)(see also, e.g., Yu, M. et al., Virology 251 (2), 227-233 (1998)).

By way of example, and not limitation, a polynucleotide encoding asoluble NiV G glycoprotein may comprise a polynucleotide sequenceencoding about amino acids 71-602 of the amino acid sequence for an NiVG Glycoprotein in Harcourt, B H et al., Virology 271: 334-349, 2000 (SEQID NO: 17) (see also Chua, K. B. et al., Science 288 (5470), 1432-1).Also by way of example, and not limitation, a polynucleotide encoding asoluble NiV G glycoprotein may comprise nucleotides 9026 to 10696 of thepolynucleotide sequence for an HeV G glycoprotein in Harcourt, B H etal., Virology 271: 334-349, 2000 (see also Chua, K. B. et al., Science288 (5470), 1432-1).

Functional equivalents of these polynucleotides are also intended to beencompassed by this invention. By way of example and not limitationfunctionally equivalent polynucleotides encode a soluble G glycoproteinof a HeV or NiV and possess one or more of the followingcharacteristics: ability to interact or bind the viral host cellreceptor, can be produced in oligomeric form or forms, the ability toelicit antibodies (including, but not limited to, viral neutralizingantibodies) capable of recognizing native G glycoprotein and/or theability to block or prevent infection of a host cell.

Polynucleotide sequences that are functionally equivalent may also beidentified by methods known in the art. A variety of sequence alignmentsoftware programs are available in the art to facilitate determinationof homology or equivalence. Non-limiting examples of these programs areBLAST family programs including BLASTN, BLASTP, BLASTX, TBLASTN, andTBLASTX (BLAST is available from the worldwide web atncbi.nlm.nih.gov/BLAST/), FastA, Compare, DotPlot, BestFit, GAP,FrameAlign, ClustalW, and PileUp. These programs are obtainedcommercially available in a comprehensive package of sequence analysissoftware such as GCG Inc.'s Wisconsin Package. Other similar analysisand alignment programs can be purchased from various providers such asDNA Star's MegAlign, or the alignment programs in GeneJockey.Alternatively, sequence analysis and alignment programs can be accessedthrough the world wide web at sites such as the CMS Molecular BiologyResource at sdsc.edu/ResTools/cmshp.html. Any sequence database thatcontains DNA or protein sequences corresponding to a gene or a segmentthereof can be used for sequence analysis. Commonly employed databasesinclude but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT,EST, STS, GSS, and HTGS.

Parameters for determining the extent of homology set forth by one ormore of the aforementioned alignment programs are well established inthe art. They include but are not limited top value, percent sequenceidentity and the percent sequence similarity. P value is the probabilitythat the alignment is produced by chance. For a single alignment, the pvalue can be calculated according to Karlin et al. (1990) Proc. Natl.Acad. Sci. (USA) 87: 2246. For multiple alignments, the p value can becalculated using a heuristic approach such as the one programmed inBLAST. Percent sequence identify is defined by the ratio of the numberof nucleotide or amino acid matches between the query sequence and theknown sequence when the two are optimally aligned. The percent sequencesimilarity is calculated in the same way as percent identity except onescores amino acids that are different but similar as positive whencalculating the percent similarity. Thus, conservative changes thatoccur frequently without altering function, such as a change from onebasic amino acid to another or a change from one hydrophobic amino acidto another are scored as if they were identical.

Polypeptides

Another aspect of this invention is directed to soluble G glycoproteinpolypeptides of HeV or NiV. The term polypeptide is used broadly hereinto include peptide or protein or fragments thereof. By way of example,and not limitation, a soluble HeV G glycoprotein may comprise aminoacids 71-604 of the amino acid sequence for a HeV G glycoprotein inWang, L. F. et al., J. Virol. 74 (21), 9972-9979 (2000) (see also, e.g.,Yu, M. et al., Virology 251 (2), 227-233 (1998)). Also by way of exampleand not limitation, a soluble NiV G glycoprotein may comprise aminoacids 71-602 of the amino acid sequence for a NiV G glycoprotein inHarcourt, B H et al., Virology 271: 334-349, 2000 (see also Chua, K. B.et al., Science 288 (5470), 1432-1).

Functional equivalents of these polypeptides are also intended to beencompassed by this invention. By way of example and not limitationfunctionally equivalent polypeptides possess one or more of thefollowing characteristics: ability to interact or bind the viral hostcell receptor, can be produced in oligomeric form or forms, the abilityto elicit antibodies (including, but not limited to, viral neutralizingantibodies) capable of recognizing native G Glycoprotein and/or theability to block or prevent infection of a host cell.

Also intended to be encompassed are peptidomimetics, which includechemically modified peptides, peptide-like molecules containingnon-naturally occurring amino acids, peptides and the like, and retainthe characteristics of the soluble G glycoprotein polypeptides providedherein. (“Burger's Medicinal Chemistry and Drug Discovery” 5th ed.,vols. 1 to 3 (ed. M. E. Wolff; Wiley Interscience 1995).

This invention further includes polypeptides or analogs thereof havingsubstantially the same function as the polypeptides of this invention.Such polypeptides include, but are not limited to, a substitution,addition or deletion mutant of the polypeptide. This invention alsoencompasses proteins or peptides that are substantially homologous tothe polypeptides. A variety of sequence alignment software programsdescribed herein above are available in the art to facilitatedetermination of homology or equivalence of any protein to a protein ofthe invention.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a polypeptide of the invention inwhich one or more residues have been conservatively substituted with afunctionally similar residue and which displays the functional aspectsof the polypeptides as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid or another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those proteins or peptideswhich contain one or more naturally-occurring amino acid derivatives ofthe twenty standard amino acids. For examples: 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a any one of the polypeptides whose sequences isdescribed herein.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polypeptide sequencein the comparison window may comprise additions or deletions (i.e. gaps)of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, ascompared to the reference sequences (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical r amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e. the windowsize) and multiplying the results by 100 to yield the percentage ofsequence identity.

Expression Vectors

This invention also relates to expression vectors comprising at leastone polynucleotide encoding a soluble G glycoprotein protein of theinvention. Expression vectors are well known in the art and include, butare not limited to viral vectors or plasmids. Viral-based vectors fordelivery of a desired polynucleotide and expression in a desired cellare well known in the art. Exemplary viral-based vehicles include, butare not limited to, recombinant retroviruses (see, e.g., PCT PublicationNos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127),alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forestvirus), Ross River virus, adeno-associated virus (AAV) vectors (see,e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655), vaccinia virus (e.g., ModifiedVaccinia virus Ankara (MVA) or fowlpox), Baculovirus recombinant systemand herpes virus.

Nonviral vectors, such as plasmids, are also well known in the art andinclude, but are not limited to, yeast and bacterial based plasmids.

Methods of introducing the vectors into a host cell and isolating andpurifying the expressed protein are also well known in the art (e.g.,Molecular Cloning: A Laboratory Manual, second edition (Sambrook, etal., 1989) Cold Spring Harbor Press). Examples of host cells include,but are not limited to, mammalian cells, such as HeLa and CHO cells.

By way of example the vector comprising the polynucleotide encoding thesoluble G protein may further comprise a tag polynucleotide sequence tofacilitate isolation and/or purification. Examples of tags include butare not limited to, myc-eptiope, S-tag, his-tag, HSV-epitope,V5-epitope, FLAG and CBP. Such tags are commercially available orreadily made by methods known to the art.

The vector may further comprise a polynucleotide sequence encoding alinker sequence. Generally the linking sequence is positioned in thevector between the soluble G protein polynucleotide sequence and thepolynucleotide tag sequence. Linking sequences can encode random aminoacids or can contain functional sites. Examples of linking sequencescontaining functional sites include but are not limited to, sequencescontaining the thrombin cleavage site or the enterokinase cleavage site.

By way of example, and not limitation, a soluble G glycoprotein may begenerated as described herein using vaccinia virus recombinants in amammalian cell culture system. Examples of primers that may be used toamplify the desired ectodomain sequence from a Hendra virus or Nipahvirus cDNA template, include, but are not limited to, the primers in theExamples.

Antibodies

Examples of antibodies encompassed by the present invention, include,but are not limited to, antibodies specific for HeV G glycoprotein,antibodies specific for NiV G glycoprotein, antibodies that cross reactwith HeV G glycoprotein and NiV G Glycoprotein and neutralizingantibodies. By way of example a characteristic of a neutralizingantibody includes, but is not limited to, the ability to block orprevent infection of a host cell. The antibodies of the invention may becharacterized using methods well known in the art.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. Preferredantibodies are derived from murine, rat, human, primate, or any otherorigin (including chimeric or humanized antibodies).

Methods of preparing monoclonal and polyclonal antibodies are well knowin the art. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired an adjuvant. Examples of adjuvants include, but are not limitedto, keyhole limpet, hemocyanin, serum albumin, bovine thryoglobulin,soybean trypsin inhibitor, Freund complete adjuvant and MPL-TDMadjuvant. The immunization protocol can be determined by one of skill inthe art.

The antibodies may alternatively be monoclonal antibodies. Monoclonalantibodies may be produced using hybridoma methods (see, e.g., Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982).

If desired, the antibody of interest may be sequenced and thepolynucleotide sequence may then be cloned into a vector for expressionor propagation. The sequence encoding the antibody of interest may bemaintained in vector in a host cell and the host cell can then beexpanded and frozen for future use. In an alternative, thepolynucleotide sequence may be used for genetic manipulation to“humanize” the antibody or to improve the affinity, or othercharacteristics of the antibody (e.g., genetically manipulate theantibody sequence to obtain greater affinity to the G glycoproteinand/or greater efficacy in inhibiting the fusion of the Hendra or Nipahvirus to the host cell receptor.).

The antibodies may also be humanized by methods known in the art. (See,for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415;5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370). In yetanother alternative, fully human antibodies may be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins.

In another alternative, antibodies may be made recombinantly andexpressed using any method known in the art. By way of example,antibodies may be made recombinantly by phage display technology. See,for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455 (1994).Alternatively, the phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro. Phage display can be performed in a variety offormats; for review see, e.g., Johnson, Kevin S, and Chiswell, David J.,Current Opinion in Structural Biology 3:564-571 (1993). By way ofexample, a soluble G glycoprotein as described herein may be used as anantigen for the purposes of isolating recombinant antibodies by thesetechniques.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65 (1995); and Pollock, et al., J Immunol Methods231:147 (1999). Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

The antibodies of the invention can be bound to a carrier byconventional methods, for use in, for example, isolating or purifyingHendra or Nipah G glycoproteins or detecting Hendra or Nipah Gglycoproteins in a biological sample or specimen. Alternatively, by wayof example, the neutralizing antibodies of the invention may beadministered as passive immunotherapy to a subject infected with orsuspected of being infected with Hendra or Nipah virus. A “subject,”includes but is not limited to humans, simians, farm animals, sportanimals and pets. Veterinary uses are also encompassed by the invention.

Diagnostics

The soluble G glycoproteins and/or antibodies of the invention may beused in a variety of immunoassays for Hendra and Nipah virus. Therecombinant expressed soluble G glycoproteins of the invention can beproduced with high quality control and are suitable as a antigen for thepurposes of detecting antibody in biological samples. By way of example,and not limitation, a soluble HEV or NiV G glycoprotein or combinationsthereof could be used as antigens in an ELISA assay to detect antibodyin a biological sample from a subject.

Vaccines

This invention also relates to vaccines for Hendra and Nipah virus. Inone aspect the vaccines are DNA based vaccines. One skilled in the artis familiar with administration of expression vectors to obtainexpression of an exogenous protein in vivo. See, e.g., U.S. Pat. Nos.6,436,908; 6,413,942; and 6,376,471. Viral-based vectors for delivery ofa desired polynucleotide and expression in a desired cell are well knownin the art and non-limiting examples are described herein. In anotheraspect, the vaccines are protein-based and comprises one or morefragments of the G protein of Hendra or Nipah virus. Preferred fragmentsare the ectodomain, and functional portions thereof, and also, portionsthat are specifically reactive to neutralizing antibodies. Portions thatare so reactive are depicted in FIG. 14. Vaccines may also beantibody-based vaccines for more immediate treatment as well asprophylaxis against infection.

Administration of expression vectors includes local or systemicadministration, including injection, oral administration, particle gunor catheterized administration, and topical administration. Targeteddelivery of therapeutic compositions containing an expression vector, orsubgenomic polynucleotides can also be used. Receptor-mediated DNAdelivery techniques are described in, for example, Findeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol.Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990)87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, andin Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

For human administration, the codons comprising the polynucleotideencoding a soluble G glycoprotein may be optimized for human use.

In another aspect of the invention, a soluble HeV or NiV G glycoproteinor combinations thereof are used as a subunit vaccine. The soluble HeVor NiV G glycoprotein or combination thereof may be administered byitself or in combination with an adjuvant. Examples of adjuvantsinclude, but are not limited, aluminum salts, water-in-soil emulsions,oil-in-water emulsions, saponin, QuilA and derivatives, iscoms,liposomes, cytokines including gamma interferon or interleukin 12, DNA,microencapsulation in a solid or semi-solid particle, Freunds completeand incomplete adjuvant or active ingredients thereof including muramyldipeptide and analogues, DEAF dextran/mineral oil, Alhydrogel, Auspharmadjuvant, and Algammulin.

The subunit vaccine comprising soluble HeV or NiV G glycoprotein orcombinations thereof can be administered orally, intravenously,subcutaneously, intraarterially, intramuscularly, intracardially,intraspinally, intrathoracically, intraperitoneally, intraventricularly,sublingually, and/or transdermally.

Dosage and schedule of administration can be determined by methods knownin the art. Efficacy of the soluble HeV or NiV G glycoprotein orcombinations thereof as a vaccine for Hendra, Nipah or relatedHenipavirus viruses may also be evaluated by methods known in the art.

Pharmaceutical Compositions

The polynucleotides, polypetides and antibodies of the invention canfurther comprise pharmaceutically acceptable carriers, excipients, orstabilizers known in the art (Remington: The Science and practice ofPharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E.Hoover.), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations, and may comprise bufferssuch as phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

The compositions used in the methods of the invention generallycomprise, by way of example and not limitation, and effective amount ofa polynucleotide or polypeptide (e.g., an amount sufficient to induce animmune response) of the invention or antibody of the invention (e.g., anamount of a neutralizing antibody sufficient to mitigate infection,alleviate a symptom of infection and/or prevent infection).

The pharmaceutical composition of the present invention can furthercomprise additional agents that serve to enhance and/or complement thedesired effect. By way of example, to enhance the immunogenicity of asoluble G polypeptide of the invention being administered as a subunitvaccine, the pharmaceutical composition may further comprise anadjuvant. Examples of adjuvants are provided herein.

Also by way of example, an not limitation, if a soluble G proteinpolypeptide of the invention is being administered to augment the immuneresponse in a subject infected with or suspected of being infected withHendra or Nipah and/or if antibodies of the present invention are beingadministered as a form of passive immunotherapy the composition canfurther comprise, for example, other therapeutic agents (e.g.,anti-viral agents)

Diagnostic Kits

The invention also provides diagnostic kits for use in the instantmethods. Kits of the invention include one or more containers comprisingby way of example, and not limitation, polynucleotides encoding asoluble G HeV or NiV G glycoprotein or combinations thereof, a soluble GHeV or NiV G glycoprotein or combinations thereof and/or antibodies ofthe invention and instructions for use in accordance with any of themethods of the invention described herein.

Generally, these instructions comprise a description of administrationor instructions for performance of an assay. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). Kits may optionally provideadditional components such as buffers and interpretive information.Normally, the kit comprises a container and a label or package insert(s)on or associated with the container.

The following examples illustrate only certain and not all embodimentsof the invention, and thus, should not be viewed as limiting the scopeof the invention.

EXAMPLES Example 1 Vector Constructs

Vectors were constructed to express transmembrane/cytoplasmictail-deleted HeV G or NiV G. The cloned cDNAs of full-length HeV or NiVG protein were amplified by PCR to generate fragments about 2600 bpencoding the transmembrane domain/cytoplasmic tail-deleted HeVG or NiV Gprotein.

The following oligonucleotide primers were synthesized for amplificationof HeV G. sHGS: 5′-GTCGACCACCATGCAAAATTACACCAGAACGACTGATAAT-3′ (SEQ IDNO 1). sHGAS: 5′-GTTTAAACGTCGACCAATCAACTCTCTGAACATTG GGCAGGTATC-3′. (SEQID NO 2).

The following oligonucleotide primers were synthesized for amplificationof NiV G. sNGS: 5′-CTCGAGCACCATGCAAAATTACACAAGATCAACAGACAA-3′ (SEQ ID NO3). sNGAS: 5′-CTCGAGTAGCAGCCGGATCAAGCTTATGTACATT GCTCTGGTATC-3′. (SEQ IDNO 4).

All PCR reactions were done using Accupol DNA polymerase (PGSScientifics Corp., Gaithersburg, Md.) with the following settings: 94°C. for 5 min initially and then 94° C. for 1 minute, 56° C. for 2minutes, 72° C. for 4 minutes; 25 cycles. These primers generated a PCRproduct for the sHeV G ORF flanked by Sal 1 sites and the sNiV G ORFflanked by Xho 1 sites. PCR products were gel purified (Qiagen,Valencia, Calif.). After gel purification, sHeV G and sNiV G weresubcloned into a TOPO vector (Invitrogen Corp., Carlsbad, Calif.).

PSectag2B (Invitrogen Corp.) was purchased and modified to contain aS-peptide tag or a myc-epitope tag. Overlapping oligonucleotides weresynthesized that encoded the sequence for the S-peptide and digested Kpn1 and EcoR1 overhangs.

SPEPS:  (SEQ ID NO 5) 5′-CAAGGAGACCGCTGCTGCTAAGTTCGAACGCCAGCACATGGATTCT-3′. SPEPAS: (SEQ ID NO 6)5′AATTAGAATCCATGTGCTGGCGTTCGAACTTAGCAGCAGCGGTCTCCT TGGTAC-3′..

Overlapping oligonucleotides were synthesized that encoded the sequencefor the myc-epitope tag and digested Kpn 1 and EcoR1 overhangs.

MTS: (SEQ ID NO 7) 5′-CGAACAAAAGCTCATCTCAGAAGAGGATCTG-3′. MTAS(SEQ ID NO 8) 5′-AATTCAGATCCTCTTCTGAGATGAGCTTTTGTTCGGTAC-3′..

64 ρmol SPEPS and 64 ρmol SPEPAS were mixed and heated to 65° C. for 5minutes and cooled slowly to 50° C. 64 ρmol MTS and 64 ρmol MTAS weremixed and heated to 65° C. for 5 minutes and cooled slowly to 50° C. Thetwo mixtures were diluted and cloned into Kpn1-EcoR1 digested pSecTag2Bto generate S-peptide modified pSecTag2B or myc-epitope modifiedpSecTag2B. All constructs were initially screened by restriction digestand further verified by sequencing.

The TOPO sG construct was digested with Sal 1 gel purified (Qiagen) andsubcloned in frame into the Xho 1 site of the S-peptide modifiedpSecTag2B or myc-epitope modified pSecTag2B. All constructs wereinitially screened by restriction digest and further verified bysequencing.

The Igκ leader-5-peptide-s HeVG (sG_(S-tag)) and the Igκ leader-myctag-sHeVG (sG_(myc-tag)) constructs were then subcloned into thevaccinia shuttle vector pMCO2 [Carroll, 1995]. Oligonucleotide SEQS:5′-TCGACCCACCATGGAGACAGACACACTCCTGCTA-3′ (SEQ ID NO 9) was synthesizedand used in combination with oligonucleotide sHGAS to amplify by PCR thesG_(S-tag) and sG_(myc-tag). All PCR reactions were done using AccupolDNA polymerase (PGS Scientifics Corp.) with the following settings: 94°C. for 5 min initially and then 94° C. for 1 minute, 56° C. for 2minutes, 72° C. for 4 minutes; 25 cycles. These primers generated PCRproducts flanked by Sal 1 sites. PCR products were gel purified(Qiagen). After gel purification, sG_(S-tag) and sG_(myc-tag) weresubcloned into a TOPO vector (Invitrogen Corp.). sG S-tag and sG myc-tagwere digested with Sal 1 and subcloned into the Sal 1 site of pMCO2. Allconstructs were initially screened by restriction digest and furtherverified by sequencing. The polypeptide structures of HeV sG S-tag andHeV sG myc-tag are depicted in a representative drawing in FIG. 6.

Example 2 Protein Production of Soluble G Protein

For protein production the genetic constructs were used to generaterecombinant poxvirus vectors (vaccinia virus, strain WR). Recombinantpoxvirus was then obtained using standard techniques employingtk-selection and GUS staining (6). Briefly, CV-1 cells were transfectedwith either pMCO2 sHeV G fusion or pMCO2 sNiV G fusion using a calciumphosphate transfection kit (Promega, Corp., Madison, Wis.). Thesemonolayers were then infected with Western Reserve (WR) wild-type strainof vaccinia virus at a multiplicity of infection (MOI) of 0.05 PFU/cell.After 2 days the cell pellets were collected as crude recombinant virusstocks. TK⁻ cells were infected with the recombinant crude stocks in thepresence of 25 μg/ml 5-Bromo-2′-deoxyuridine (BrdU) (Calbiochem, LaJolla, Calif.). After 2 hours the virus was replaced with an EMEM-10overlay containing 1% low melting point (LMP) agarose (LifeTechnologies, Gaithersburg, Md.) and 25 μg/ml BrdU. After 2 days ofincubation an additional EMEM-10 overlay containing 1% LMP agarose, 25μg/ml BrdU, and 0.2 mg/ml 5-Bromo-4-chloro-3-indolyl-β-D-glucuronic acid(X-GLUC) (Clontech, Palo Alto, Calif.) was added. Within 24-48 hoursblue plaques were evident, picked and subject to two more rounds ofdouble selection plaque purification. The recombinant vaccinia virusesvKB16 (sHeV G fusion) and vKB22 (sNiV G fusion) were then amplified andpurified by standard methods. Briefly, recombinant vaccinia viruses arepurified by plaque purification, cell-culture amplification, sucrosecushion pelleting in an ultracentrifuge and titration by plaque assay.Expression of sHeV G was verified in cell lysates and culturesupernatants (FIG. 1).

As shown in FIG. 1, vaccinia virus encoding either a myc-tag or S-tagsoluble HeV G was produced by metabolic labeling in HeLa cells. Controlis wild-type HeV G. Specific precipitation of each sG construct is shownby precipitation from either lysates or supernatants using myc MAb orS-beads.

Example 3 Properties of Soluble G Protein

To demonstrate that the recombinant expressed, soluble, purified G (sHeVG) retained desirable properties (e.g. native structural features suchas receptor binding competence), it has been demonstrated thatpre-incubation of target cells with affinity-purified sHeV G results ina dose-dependent inhibition of virus-mediated fusion in severaldifferent cell lines that are susceptible to virus-mediated fusion andinfection (FIG. 2).

For purification of soluble G glycoproteins, HeLa cells were infectedwith vKB15 or vKB16 (moi=3) for 2 hours. After infection the virus wasremoved and serum-free OptiMem medium (Invitrogen, Corp.) was added.After 36 hours, the supernatants were removed and clarified bycentrifugation. A S-protein column was poured with 15 ml of S-proteinagarose (Novagen) in a XK26 column (Amersham Pharmacia Biotech,Piscataway, N.J.). The S-protein column was washed with 10 bed volumesof PBS. The supernatant from vKB16-infected cells was passed overS-protein agarose column, the column was washed with 10 bed volumes ofPBS, and the sG_(S-tag) was eluted with 1 bed volume of 0.2M citratepH=2 into 20 ml 1M Tris pH=8. Lentil lectin Sepharose B was purchased(Amersham Pharmacia Biotech) and a 25 ml XK26 column was poured. Thesupernatants from vKB15-infected cells were passed over the lentillectin column, the column was washed with 10 bed volumes PBS, and thesG_(myc-tag) was eluted with 1 bed volume of 0.2M glycine pH=2.5 into 2ml 1M Tris pH=8. Both eluates were then concentrated using 30 kDACentricon centrifugal filter units (Millipore, Billerica, Mass.) andfilter sterilized. Protein concentrations were calculated usingSDS/PAGE, Commassie brilliant blue R-250 staining and densitometryanalysis with NIH image 1.62 software.

As shown in FIG. 2, soluble HeV G envelope glycoprotein (either theS-tag or myc-tag versions) blocks both HeV and NiV-mediated cell-cellfusion. Dose response inhibition of HeV and NiV cell-cell fusion wasconducted by pre-incubating target cells with the indicated amount ofpurified sG for 30 min at room temperature. Effector cells expressingeither HeV or NiV F and G were added and fusion was allowed to proceedfor 2.5 h at 37° C. Reaction mixes were processed for β-gal productionusing the β-gal reporter gene assay. Assays were carried out induplicate. Panels A and B show that crude sHeV G (S-tag) containingsupernatant can potently block HeV-mediated fusion in two alternant celltypes, while a control supernatant from WR infected cells(non-recombinant vaccinia virus infected) has no effect. Panels C and D:Inhibition of HeV and NiV-mediated fusion by sG S-tag in U373 cells(Panel C) or PCI 13 cells (Panel D). Panels E and F: Inhibition of HeV-and NiV-mediated fusion by purified sG myc-tag in U373 cells (Panel E)or PCI 31 cells (Panel F).

As additional evidence, indirect immunofluorescence was performed thatdemonstrated sHeV G can specifically bind to cell lines that aresusceptible to virus-mediated fusion and infection (FIG. 3). sHeV G isunable to bind to HeLa cells, a non-permissive cell line for HeV andNiV-mediated fusion and virus infection. These data suggest that sHeV Ginhibits HeV-mediated fusion by binding to the putative receptor ontarget cells thus blocking subsequent attachment and fusion of HeV G andF expressing effector cells. The interaction of sHeV G with the putativeHeV receptor may be a useful tool for receptor purification andidentification. Since the soluble G glycoprotein can be expressed andpurified and also exhibits biochemical features similar to that whatwould be expected from the native G glycoprotein making it an idealsubunit immunogen for the elicitation of virus-neutralizing antibodies.A similar sG construct has been made using the same S-tag approach (seemethods above) using the coding sequence of the G envelope glycoproteinof Nipah virus. This sNiV G (S-tag) has been cloned and expressed and isshown in FIG. 4.

A final analysis of the sHeV G (S-tag) envelope glycoprotein was made toevaluate the predicted oligomeric nature of the protein. The retentionof some oligomeric properties of a soluble and secreted G glycoproteinmay be important in retaining critical immunological or biochemicalfeatures as discussed in the introduction above. FIG. 5 shows ananalysis of secreted sHeV G (S-tag) glycoprotein by sucrose gradientfractionation which identifies monomeric, dimeric and tetrameric formsof the G glycoprotein. Both cross-linked and non-cross-linked materialswere analyzed. The results indicate that monomeric, dimeric, and sometetrameric sG comprises the sG preparation, and this is in agreementwith findings on soluble and full-length versions of other paramyxovirusH and HN attachment glycoproteins (discussed above). These three speciescould be separated by preparative size exclusion chromatographytechniques if desired.

Example 4 Characterization of Soluble and Secreted HeV G

It was next sought to determine if the secreted sG was oligomeric innature. The apparent molecular weight of purified sG material was firstexamined using size exclusion chromatography with a calibrated Superdex200 analytical grade column 10/300. A 500 μg aliquot of eithersG_(S-tag) or sG_(myc-tag) was passed over the Superdex 200 andfractions were collected using the same methods employed for the highmolecular weight standards. Essentially identical results were observedwith both the sG_(S-tag) and sG_(myc-tag) glycoproteins, and the resultsshown in FIG. 7 are those for sG_(S-tag). The locations of the proteinstandards and the three major species of sG are indicated in the figure.The inset shows the profile of separated sG. The analysis of purifiedsG_(S-tag) in seven independent separation experiments indicated twomajor peaks with apparent molecular weights of ˜372 KDa+/−19 KDa (˜60%of the material) and ˜261 KDa+/−47 KDa (˜35% of the material), and oneminor peak of ˜741 KDa+/−40 KDa (˜5% of the material). These resultsindicated that at least some of the material would be oligomeric innature, consistent with the glycoprotein's expected structure. However,from prior experience in the preparation and analysis of solublevirus-derived membrane glycoproteins, such as gp120 from HIV-1, suchmolecular weight calculations derived from size-exclusion chromatographyanalysis may be over-estimated.

To further characterize the apparent oligomeric species of sG,sG_(S-tag) was analyzed using sucrose gradient densitometry. For thisanalysis, the sG_(S-tag) glycoprotein was chosen because it can beaffinity-precipitated with S-protein agarose circumventing the need forspecific MAb. FIG. 8 depicts the oligomeric profiles of metabolicallylabeled sG_(S-tag) isolated from the supernatant of expressing cells.Following brief centrifugation to remove any cellular debris thesupernatant was concentrated and buffer replaced with PBS as describedherein. Prior to separation in the sucrose gradient, half of thesupernatant was cross-linked with DTSSP, a reducible cross-linker, anduncross-linked and cross-linked material were loaded onto two separatesucrose gradients. After fractionation of each gradient, the fractionswere split into 2 tubes, precipitated with S-protein agarose, washed andresuspended in SDS sample buffer, one set with and one set withoutβ-mercaptoethanol and all four sets of fractions then analyzed bySDS-PAGE. FIGS. 8A and 8B are uncross-linked sG_(S-tag) separated on thesucrose gradient, fractionated, and immunoprecipitated. In FIG. 8A, thefractions were resolved on SDS-PAGE in the absence of β-mercaptoethanol,whereas in FIG. 8B, the fractions were resolved on SDS-PAGE in thepresence of β-mercaptoethanol. FIGS. 8C and 8D are cross-linkedsG_(S-tag) separated on the sucrose gradient, fractionated, andimmunoprecipitated. In FIG. 8C, the fractions were resolved on SDS-PAGEin the absence of β-mercaptoethanol whereas in FIG. 8D, the fractionswere resolved on SDS-PAGE in the presence of β-mercaptoethanol. Thestarting material for each gradient was also run on each gel in thepresence or absence β-mercaptoethanol and is illustrated as control.From the data shown in FIGS. 8A and 8C, it was determined that for boththe uncross-linked and cross-linked sG_(S-tag), there are three distinctspecies of sG present. Based on the apparent molecular weights of sG ineach of the fractions across each of the gradients, these three specieslikely represent monomer, dimer and tetramer. In addition, the immediatecross-linking of the sG with DTSSP prior to gradient centrifugation didnot significantly increase the amount of either the tetrameric ordimeric species. The analysis of the non-cross-linked, un-reduced andreduced sG clearly indicates that the dimeric oligomer is disulfidelinked, which was anticipated based on data derived from otherparamyxovirus attachment glycoproteins (36, 44). The dimers of otherparamyxovirus attachment glycoproteins have been shown to form atetramer on the surface of infected cells, and it is generally believedthat the native oligomeric structure is a dimer of dimers. To analyzethis possibility here, full-length HeV G was expressed and metabolicallylabeled in HeLa cells (a receptor-negative cell line) and performed asimilar experiment and sucrose gradient analysis. Following either across-linking procedure, or no treatment, of surface-expressed HeV G onintact cells, the cells were lysed with non-ionic detergent, lysatesclarified by centrifugation, and the surface-expressed HeV Gpreparations analyzed by sucrose gradient centrifugation. Fractions wereanalyzed by immunoprecipitation with polyclonal anti-HeV rabbit serafollowed by Protein G-Sepharose and resolved on SDS-PAGE under reducingand non-reducing conditions as before. Shown in FIG. 9 is sucrosegradient analysis of surface expressed uncross-linked full lengthradiolabeled HeV G. Here it is observed that in the non-reducedfractions, >95% of the full-length HeV G exists as the apparenttetrameric species (FIG. 9A, lanes 2-5) and this oligomeric species isclearly dependent on disulfide bonds as illustrated by the correspondingreduced fractions which are monomeric (FIG. 9B, lanes 2-5). In addition,identical sucrose gradient profiles were seen regardless of whethercross-linking reagent was used or not indicating that the nativecell-surface expressed G forms a very stable tetrameric oligomer. Theprotein's natural membrane anchor domain and or its cytoplasmic tail maycontribute to this stability. Nevertheless, the majority (˜60-70%) ofthe sG_(s-tag) glycoprotein product produced here is an oligomeric dimerwhich indicates that it may retain important and useful nativestructural features.

Example 5 Inhibition of HeV and NiV Infection by Soluble HeV G

It was next evaluated if sG_(S-tag) effects on live virus infection ofVero cells in culture. Here, following preincubation of Vero cells withvarious concentrations of sG_(s-tag), the cells were infected with1.5×10³ TCID₅₀/ml and 7.5×10² TCID₅₀/ml of live HeV or NiV,respectively, in the presence of sG_(S-tag) for 30 min, followed byremoval of the virus inoculum and incubation with sG_(S-tag). After 24hrs in culture, the number of HeV and NiV infection foci was quantifiedby specific immunostaining of cell monolayers with ananti-phosphoprotein (P) as detailed in the methods. Representativeexamples of infected Vero cells in the presence or absence of sG_(S-tag)are shown in FIG. 10. Typically, infection of Vero cells with live HeVor NiV produces characteristic syncytium morphologies for each virus.Immunofluorescence for HeV P protein in HeV syncytia demonstrated thatHeV reproducibly infects and incorporates surrounding cells into eachsyncytium with cell nuclei and viral protein equally detectablethroughout the majority of infected cells (FIG. 10A). NiV infected cellsinitially show a similar appearance to HeV syncytia, but ultimatelyincorporated nuclei within each giant cell are sequestered togethertowards the periphery while the remaining cellular debris is alsoarranged around the outside leaving the central region largely empty.Thus, immunofluorescence for HeV P protein in NiV syncytia often appearas hollow spheres coated in viral antigen (FIG. 10B) and by comparison,the untreated control HeV infections produce smaller syncytium relativeto the untreated NiV control (FIGS. 10A and 10B). FIGS. 10C and 10D arerepresentative examples of HeV and NiV-infected Vero cells in thepresence of 100 μg/ml sG_(S-tag). Although there were still someinfected cells present as detected by immunofluorescence, syncytiaformation was completely blocked in both HeV and NiV-infected cells(FIGS. 10C and 10D, respectively). Furthermore, quantitative analysis ofthe inhibition of HeV and NiV infection by purified sG_(S-tag)glycoprotein revealed a dose-dependent response, further demonstratingits specificity, as shown in FIG. 11. Together these data provide strongevidence that HeV and NiV utilize a common receptor on the surface ofthe host cell. Additionally, the specific inhibition of both viruses bysG_(S-tag) further demonstrated that the sG_(S-tag) construct maintainsimportant native structural elements. Interestingly, HeV infection wasinhibited significantly better than NiV such that the IC₅₀ determinedfor sG_(S-tag) was four-fold greater for NiV (13.20 μg/ml) than for HeV(3.3 μg/ml) (FIG. 11). Given the current evidence suggesting bothviruses utilize a common receptor, the reasons for the differencesobserved in sG_(S-tag) inhibition of virus infection versus cell-fusionremain unknown. A similar difference in the ability of sG_(S-tag) toinhibit HeV and NiV-mediated cell-fusion was not observed, asdemonstrated in FIG. 3. Although HeV-mediated fusion was more potentthan NiV-mediated fusion, illustrated by the higher levels of substrateturnover, the sG_(S-tag) IC₅₀ in both cell-fusion assays remainedconstant. In previous reports, it has been demonstrated throughheterotypic function that the difference in cell-fusion rates betweenHeV and NiV was dependent on the fusion protein. Here, it isdemonstrated that natural NiV infection appears to be more vigorous thanHeV infection. Perhaps other viral proteins present during infection areinfluencing the kinetics of infection thus altering the inhibitionsusceptibility, or they may be differences in the affinity of HeV sGversus NiV G to the cell surface expressed receptor.

Example 6 Soluble HeV G Elicits a Potent Virus-Neutralizing PolyclonalAntibody Response

With few exceptions, it is the envelope glycoproteins of viruses towhich virtually all neutralizing antibodies are directed and allsuccessful human viral vaccines induce neutralizing antibodies that cancross-react with immunologically relevant strains of a virus. Morespecifically, virus-neutralizing antibodies are the key vaccine-inducedprotective mechanism in the case of the paramyxoviruses mumps andmeasles, and it has been shown that vaccinia virus expressed full-lengthenvelope glycoproteins from NiV can elicit virus-neutralizingantibodies. Data indicate that the sG_(S-tag) glycoprotein retainsimportant structural features based on its abilities to specificallybind receptor positive cells and block both HeV and NiV-mediated fusionand infection. Thus, the immunization of animals with sG shouldpotentially generate potent virus-neutralizing antibodies. To test thispossibility, purified sG_(S-tag) was used to immunize rabbits and theresulting anti-G antiserum evaluated in virus neutralization assays withboth HeV and NiV. Table 1 summarizes the neutralization of HeV and NiVinfection by the polyclonal rabbit anti-G sera. The sera from bothrabbits were capable of complete neutralization of HeV at a dilution of1:1280. NiV was also neutralized by the sG_(S-tag) antiserum, withcomplete neutralization at a dilution of 1:640. A two fold difference intiter is consistent with partial antibody cross-reactivity of the HeVand NiV G glycoproteins. Pre-bleeds from both rabbits were also testedfor their ability to neutralize HeV and NiV. Although there was slightneutralization at the highest concentration, this activity wascompletely abrogated upon dilution of the sera. Previous studies havedemonstrated that HeV and NiV antisera do cross neutralize, with eachserum being slightly less effective against the heterotypic virus (14).Moreover, it has been demonstrated a similar trend incross-neutralization using the cell-fusion assay for HeV and NiV (4,81).Because sG_(S-tag) was able to elicit such a potent immune response withhigh levels of neutralizing antibodies, it may provide an avenue forvaccine development strategies.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims.

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TABLE 1 HeV NiV Dilution Rabbit 405 Rabbit 406 Rabbit 405 Rabbit 4061:10 − − − − − − − − 1:20 − − − − − − − − 1:40 − − − − − − − − 1:80 − −− − − − − − 1:160 − − − − − − − − 1:320 − − − − − − − − 1:640 − − − − −− − − 1:1,280 − − − − + + − + 1:2,560 − − − + + + + + 1:5,120 −− + + + + + + 1:10,240 + + + + + + + + 1:20,480 + + + + + + + +

The invention claimed is:
 1. A monoclonal antibody specifically reactiveto a polypeptide comprising a soluble form of the ectodomain of Hendravirus G protein.
 2. A vaccine for preventing or treating infection of apatient by Hendra virus comprising the antibody of claim
 1. 3. A methodof detecting infection by a Hendra virus comprising detecting Hendravirus antigens or antibodies to Hendra virus in a sample obtained from asubject suspected of being infected using the monoclonal antibody ofclaim 1.